Validation of a liquid chromatography‐tandem mass spectrometry method for simultaneous quantification of N,N‐dimethylacetamide and N‐monomethylacetamide in pediatric plasma

Abstract

N,N–dimethylacetamide is an excipient used in intravenous busulfan formulations, a drug used in hematopoietic stem cell transplantation conditioning. The aim of this study was to develop and validate a liquid chromatography‐tandem mass spectrometry method for simultaneous quantification of N,N‐dimethylacetamide, and its metabolite N‐monomethylacetamide in plasma from children receiving busulfan. A 4 μl aliquot of patient plasma was extracted using 196 μl 50% methanol solution and quantified against calibrators prepared in the extraction solvent given negligible matrix effects across three concentrations. 9[H2]‐N,N‐dimethylacetamide was used as an internal standard. Separation of N,N‐dimethylacetamide and N‐monomethylacetamide was achieved using a Kinetex EVO C18 stationary phase (100 mm × 2.1 mm × 2.6 μm) running an isocratic mobile phase of 30% methanol containing 0.1% formic acid at a flow of 0.2 ml/min over 3.0 min. The injection volume was 1 μl. Calibration curves for N,N‐dimethylacetamide and N‐monomethylacetamide were linear up to 1200 and 200 μg/L, respectively, with a lower limit of quantification 1 μg/L for both analytes. Calibrator accuracy and precision were within ± 10% of the test parameters across four concentration levels. Analytes were stable over 14 days at three different storage conditions. This method was successfully applied to measure N,N‐dimethylacetamide and N‐monomethylacetamide concentrations in a total of 1265 plasma samples from 77 children.